1. Field of the Invention
The present invention relates to a recombinant antibody and an antibody fragment thereof which specifically react with the extracellular domain of human CC chemokine receptor 4 (hereinafter referred to as “CCR4”). Furthermore, the present invention relates to a recombinant antibody such as humanized antibody, human antibody and the like, and an antibody fragment thereof which specifically react with CCR4, have cytotoxic activity and activity of inhibiting production of cytokine by Th2 cells, and comprise a specific complementarity determining region (hereinafter referred to as “CDR”). Moreover, the present invention relates to a DNA encoding the above mentioned antibody. Also, the present invention relates to a vector comprising the DNA, and a transformant transformed with the vector. Still furthermore, the present invention relates to a method for producing the above mentioned antibody using the transformant, and a medicament such as a therapeutic agent, a diagnostic agent and the like, for Th2-mediated immune diseases such as allergic diseases and the like, which comprises using the antibody. Still moreover, the present invention relates to a medicament such as a therapeutic agent, a diagnostic agent and the like, for cancers such as blood cancers, e.g., leukemia, which comprises using the antibody.
2. Brief Description of the Background Art
Various factors such as eosinophils, mast cells, IgE and the like, relate to allergic diseases such as bronchial asthma. Eosinophils infiltrate into an inflammatory part, release a cytotoxic basic protein such as MBP (major basic protein) or the like, by degranulation to thereby induce injury of surrounding tissues. Mast cells release histamine by binding to an antigen immune complex with IgE produced from B cells to thereby induce an immediate allergic reaction. They are controlled by biologically functional molecules such as cytokine, chemokine, and the like, which take part in signal transduction between cells. Eosinophils are subjected to differentiation induction and life span prolongation by IL-5, and degranulation is further induced. IgE is produced from B cells activated by IL-4 and becomes an immune complex with the antigen to accelerate degranulation of mast cells. It has been found that IL-4, IL-13 and the like are also produced from mast cells and contribute to the production of IgE from B cells, and the presence of an allergy reinforcing loop has been confirmed (Am. J. Respir. Crit. Care Med., 152: 2059 (1995), Immunol. Today, 15: 19 (1994)). Thus, an elaborately cytokine-chemokine network is present between inflammatory cells and keeps complicated balances.
The cytokine and chemokine are produced by helper T cells which express CD4 on the cell surface (hereinafter referred to as “CD4+ Th cells”). Actually, it has been found that infiltration of helper T cells is remarkably found in the airway inflammation part of bronchial asthma patients, wherein a considerably large number of the T cells are activated and that the degree of seriousness and airway hypersensitivity of asthma correlates with the number of activated T cells, as well as the activated T cells are also increased in the peripheral blood (Am. Rev. Respir. Dis., 145: S22 (1992)).
The helper T cells are classified into Th1 cells and Th2 cells, depending on the kind of cytokine to be produced thereby (Annu. Rev. Immunol., 7: 145 (1989)). Examples of the cytokine produced by Th2 cells include IL-4, IL-5, IL-13, and the like.
It has been found that an antigen-specific T cell clone isolated from an atopic disease patient releases Th2 cytokine when stimulated in vitro (Proc. Natl. Acad. Sci., U.S.A., 88: 4538 (1991)), and Th2 cells are present in bronchoalveolar lavage fluid (hereinafter referred to as “BAL”) and airway mucosa of asthma patients (N. Engl. J. Med., 326: 298 (1992), Eur. J. Immunol., 23: 1445 (1993)). IL-4 and IL-5 of Th2 cytokines are increased when the expression of mRNA in BAL is examined using an allergic inflammation animal model (Clin. Immunol. Immunopathol., 75: 75 (1995)). Also, when induced Th2 cells in mice are administered into the vein and nasal cavity, an antigen-specific asthmatoid inflammatory symptom is induced in the lungs (J. Exp. Med., 186: 1737 (1997), J. Immunol., 160: 1378 (1998)) and causes eosinophilia (J. Immunol., 161: 3128 (1998)). Expression of IL-5 is observed in the airway mucous membrane of asthma patients and the lesions of atopic dermatitis patients (J. Clin. Invest., 87: 1541 (1991), J. Exp. Med., 173: 775 (1991)), and the expression level of IL-13 in the mucous membrane of continuous allergic rhinitis well correlates with the amounts of serum total IgE and antigen-specific IgE (Therapeutics, 32: 19 (1998)).
Chemokine is a general term for basic heparin-binding proteins having leukocyte migration and leukocyte activation functions, and classified into subfamilies of CXC, CC, C and CX3C depending on the position of the cysteine residue preserved on the primary structure. Up to date, 16 kinds of chemokine receptors have been identified (Curr. Opi. Immunol., 11: 626 (1999)), and it has been shown that expression of each chemokine receptor is different on the surface of each leukocyte such as Th1 cell, Th2 cell or the like (Cell Engineering, 17: 1022 (1998)).
Human CCR4 is a G protein complexed seven transmembrane receptor cloned as K5-5 from a human immature basophilic cell line KU-812, and has the amino acid sequence represented by SEQ ID NO:17. Since the transmembrane regions of CCR4 are considered to be positions 40-67, positions 78-97, positions 113-133, positions 151-175, positions 207-226, positions 243-270 and positions 285-308, it is considered that the extracellular domains are positions 1-39, positions 98-112, positions 176-206 and positions 271-284 in the amino acid sequence, and that the intracellular regions are positions 68-77, positions 134-150, positions 227-242 and positions 309-360 (J. Biol. Chem., 270: 19495 (1995)). When cloning was carried out, it was reported that the ligand of CCR4 is MIP-1α (macrophage inflammatory protein-1α), RANTES (regulated on activation normal T-cell expressed and secreted) or MCP-1 (monocyte chemotactic protein) (Biochem. Biophys. Res. Commun., 218: 337 (1996), WO 96/23068). However, thereafter, it has been found that TARC (thymus and activation-regulated chemokine) produced from stimulated human peripheral blood mononuclear cells (hereinafter referred to as “PBMC”) and thymus cells (J. Biol. Chem., 271: 21514 (1996)) specifically binds to CCR4 (J. Biol. Chem., 272: 15036 (1997)). It has been also reported that MDC (macrophage-derived chemokine) isolated from macrophage (J. Exp. Med., 185: 1595 (1997)), also known as STCP-1 (stimulated T cell chemotactic protein-1) (J. Biol. Chem., 272: 25229 (1997)), bind to CCR4 more strongly than TARC (J. Biol. Chem., 273: 1764 (1998)).
It has been shown that CCR4 is expressed on CD4+ Th cells which produce cytokine and/or chemokine (J. Biol. Chem., 272: 15036 (1997)), and it has been reported that CCR4 is expressed selectively on Th2 cells among CD4+ Th cells (J. Exp. Med., 187: 129 (1998), J. Immunol., 161: 5111 (1998)). In addition, CCR4+ cells have been found in a group of effector/memory T cells (CD4+/CD45RO+), and when CCR4+ cells were stimulated, IL-4 and IL-5 were produced but IFN-γ was not produced (Int. Immunol., 11: 81 (1999)). Also, it has been reported that CCR4+ cells belong to a CLA (cutaneous lymphocyte antigen)-positive and α4β8 integrin-negative group among memory T cells, and CCR4 is expressed on memory T cells related not to gut immunity but to systemic immunity of the skin and the like (Nature, 400: 776 (1999)). These results strongly suggest a possibility that the memory T cells which are activated by induction of inflammation express CCR4, migrate into the inflammatory site by ligands, MDC and TARC, and accelerate activation of other inflammatory cells.
As the current method for treating Th2-mediated immune diseases, the followings have been developed: (1) antagonists for cytokine and chemokine such as a humanized anti-IL-5 antibody (SB-240563: Smith Kline Beecham, Sch-55700 (CDP-835): Shehling Plaw/Celltech), a humanized IL-4 antibody (U.S. Pat. No. 5,914,110), a soluble chemokine receptor (J. Immunol., 160: 624 (1998)), etc.; (2) cytokine chemokine production inhibitors such as an IL-5 production inhibitor (Japanese Published Unexamined Patent Application No. 53355/96), a retinoid antagonist (WO 99/24024), splatast tosilate (IPD-1151T, manufactured by Taiho Pharmaceutical), etc.; (3) agents for eosinophil, mast cell and the like as final inflammatory cells, such as a humanized IL-5 receptor antibody (WO 97/10354), a CC chemokine receptor 3 (CCR3) antagonist (Japanese Published Unexamined Patent Application No. 147872/99), etc.; (4) inflammatory molecule inhibitors such as a humanized anti-IgE antibody (Am. J. Respir. Crit. Care Med., 157: 1429 (1998)), etc.; and the like. But they inhibit only a part of the elaborate network among cytokine, chemokine and inflammatory cells and are therefore not radical. Anti-CD4 antibodies have an ability to control T cells, and have effects on serious steroid-dependent asthma. However, since the CD4 molecule is broadly expressed in various immune cells, they lack in specificity and have a drawback of accompanying strong immunosuppressive effect (Int. Arch. Aller. Immunol., 118: 133 (1999)).
Thus, in order to inhibit all of them, it is required to control specifically upstream of the problematic allergic reaction, namely Th2 cells.
The currently used principal method for treating patients of serious Th2-mediated immune diseases is steroid administration, but side effects by steroids cannot be avoided. Also, there are drawbacks that the conditions of each patient return to the original pathology when the steroid administration is suspended, and that drug resistance is acquired when the steroid is administered for a long time.
To date, no monoclonal antibody which can detect CCR4-expressing cells and also has cytotoxicity against CCR4-expressing cells has been established. In addition, no therapeutic agent which can inhibit production of Th2 cytokine has been known so far.
Although it has been reported that CCR4 is also expressed on leukemia (Blood, 96: 685 (2000)), no therapeutic agent which injures leukemia cells has been known.
It is known in general that when an antibody derived from a non-human animal, e.g., a mouse antibody, is administered to human, it is recognized as an foreign substance and induces a human antibody against the mouse antibody (human anti-mouse antibody: hereinafter referred to as “HAMA”) in the human body. It is known that the HAMA reacts with the administered mouse antibody to cause side effects (J. Clin. Oncol., 2: 881 (1984), Blood, 65: 1349 (1985), J. Natl. Cancer Inst., 80: 932 (1988), Proc. Natl. Acad. Sci. U.S.A., 82: 1242 (1985)), to quicken disappearance of the administered mouse antibody from the body (J. Nucl. Med., 26: 1011 (1985), Blood, 65: 1349 (1985), J. Natl. Cancer Inst., 80: 937 (1988)), and to reduce therapeutic effects of the mouse antibody (J. Immunol., 135: 1530 (1985), Cancer Res., 46: 6489 (1986)).
In order to solve these problems, attempts have been made to convert an antibody derived from a non-human animal into a humanized antibody such as a human chimeric antibody, a human complementarity determining region (hereinafter referred to as “CDR”)-grafted antibody or the like using genetic engineering technique. The human chimeric antibody is an antibody in which its antibody variable region (hereinafter referred to as “V region”) is an antibody derived from a non-human animal and its constant region (hereinafter referred to as “C region”) is derived from a human antibody (Proc. Natl. Acad. Sci. U.S.A., 81: 6851 (1984)). The human CDR-grafted antibody is an antibody in which the amino acid sequence of CDR in the V region derived from a non-human animal antibody is grafted into an appropriate position of a human antibody (Nature, 321: 522 (1986)). In comparison with antibodies derived from non-human animals such as mouse antibodies and the like, these humanized antibodies have various advantages for clinical applications. For example, regarding immunogenicity and stability in blood, it has been reported that blood half-life of a human chimeric antibody became about 6 times as long as a mouse antibody when administered to human (Proc. Natl. Acad. Sci. U.S.A., 86: 4220 (1989)). In the case of a human CDR-grafted antibody, it has been reported that its immunogenicity was reduced and its serum half-life became 4 to 5 times as long as a mouse antibody in experiment using a monkey (J. Immunol., 147: 1352 (1991)). Thus, it is expected that the humanized antibodies have less side effects and their therapeutic effects continue for a longer time than antibodies derived from non-human animals. Also, in treatment particularly for reducing the number of CCR4-expressing cells, higher cytotoxic activity such as complement-dependent cytotoxic activity (hereinafter referred to as “CDC activity”), antibody-dependent cell-mediated cytotoxic activity (hereinafter referred to as “ADCC activity”) and the like, via the Fc region (the region in and after the hinge region of an antibody heavy chain) of an antibody are important for the therapeutic effects. It has been reported that on such cytotoxic activities, the human antibodies are superior to antibodies derived from non-human animals since the Fc region of human antibodies more efficiently activates human complement components and human effector cells having Fc receptor on the cell surface such as monocytes, macrophages, NK cells, and the like, than the Fc region of antibodies derived from non-human animals. For example, it has been reported that tumor cytotoxic activity by human effector cells was increased in a human chimeric antibody prepared by converting the Fc region of an mouse antibody for GD2 into the Fc region of a human antibody (J. Immunol., 144: 1382 (1990)), and similar results have also been reported on a human CDR-grafted antibody for CAMPATH-1 antigen (Nature, 332: 323 (1988)).
These results clearly show that humanized antibodies are preferred to antibodies derived from non-human animals such as mouse antibodies and the like, as antibodies for clinical applications to human.
Furthermore, according to the recent advances in protein engineering and genetic engineering, antibody fragments having a smaller molecular weight such as Fab, Fab′, F(ab′)2, a single chain antibody (hereinafter referred to as “scFv”) (Science, 242: 423 (1988)), a disulfide stabilized V region fragment (hereinafter referred to as “dsFv”) (Molecular Immunol., 32: 249 (1995)) and the like, can be produced. Since the fragments are smaller in molecular weight than the complete antibody molecules, they are excellent in transitional ability into target tissues (Cancer Res., 52: 3402 (1992)). It is considered that these fragments derived from humanized antibodies are more desirable than those derived from antibodies derived from non-human animals such as mouse antibodies, when used in clinical applications to human.
As described above, diagnostic and therapeutic effects can be expected from humanized antibodies and fragments thereof when used alone, but attempts have been made to further improve the effects by using other molecules in combination. For example, cytokine can be used as one of such molecules. Cytokine is a general term for various soluble factors which control intercellular mutual functions in immune reactions. CDC activity and ADCC activity, for example, are known as the cytotoxic activities of antibodies, and ADCC activity is controlled by effector cells having Fc receptors on the cell surface such as monocytes, macrophages, NK cells and the like (J. Immunol., 138: 1992 (1987)). Since various cytokines activate these effector cells, they can be administered in combination with an antibody in order to improve ADCC activity of the antibody and the like.